Supercontracted state of vertebrate smooth muscle cell fragments reveals myofilament lengths.
Isolated cell preparations from chicken gizzard smooth muscle typically contain a mixture of cell fragments and whole cells. Both species are spontaneously permeable and may be preloaded with externally applied phalloidin and antibodies and then induced to contract with Mg ATP. Labeling with antibodies revealed that the cell fragments specifically lacked certain cytoskeletal proteins (vinculin, filamin) and were depleted to various degrees in others (desmin, alpha-actinin). The cell fragments showed a unique mode of supercontraction that involved the protrusion of actin filaments through the cell surface during the terminal phase of shortening. In the presence of dextran, to minimize protein loss, the supercontracted products were star-like in form, comprising long actin bundles radiating in all directions from a central core containing myosin, desmin, and alpha-actinin. It is concluded that supercontraction is facilitated by an effective uncoupling of the contractile apparatus from the cytoskeleton, due to partial degradation of the latter, which allows unhindered sliding of actin over myosin. Homogenization of the cell fragments before or after supercontraction produced linear bipolar dimer structures composed of two oppositely polarized bundles of actin flanking a central bundle of myosin filaments. Actin filaments were shown to extend the whole length of the bundles and their length averaged integral to 4.5 microns. Myosin filaments in the supercontracted dimers averaged 1.6 microns in length. The results, showing for the first time the high actin to myosin filament length ratio in smooth muscle are readily consistent with the slow speed of shortening of this tissue. Other implications of the results are also discussed.
Small
J V
JV
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg.
Herzog
M
M
Barth
M
M
Draeger
A
A
D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000199:Actins / Q000032:analysis; D000818:Animals; D002645:Chickens; D003598:Cytoskeletal Proteins / Q000032:analysis; D005895:Gizzard, Avian / Q000166:cytology / Q000648:ultrastructure; D015151:Immunoblotting; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D009130:Muscle, Smooth / Q000166:cytology / Q000648:ultrastructure*
D000199:Actins; D003598:Cytoskeletal Proteins